Detoxified mutants of bacterial ADP-ribosylating toxins as parenteral adjuvants

ABSTRACT

The present invention provides parenteral adjuvants comprising detoxified mutants of bacterial ADP-ribosylating toxins, particularly those from pertussis (PT), cholera (CT), and heat-labile  E. coli  (LT).

CROSS-REFERENCE TO RELATED APPLICATION

This application is related to provisional patent application Ser. No.60/041,227, filed Mar. 21, 1997, from which priority is claimed under 35USC §119(e) (1) and which is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to adjuvants useful for the administrationof antigens to organisms. In particular, the adjuvants of the inventionallow the parenteral delivery of vaccines to raise an immune response.

BACKGROUND OF THE INVENTION

Advances in recombinant DNA technology have made possible the generationof a variety of vaccines, such as subunit vaccines and DNA-basedvaccines. These are in addition to the more traditional killed orattenuated vaccines. Adjuvants that enhance the immune system's responseto antigenic material are known; however, currently, few adjuvants areapproved for human usage, although additional adjuvants are inpre-clinical and clinical studies.

The ADP-ribosylating bacterial toxins, a group of potent toxins tohumans, include diphtheria toxin, pertussis toxin (PT), cholera toxin(CT), the E. coli heat-labile toxins (LT1 and LT2), Pseudomonasendotoxin A, C. botulinum C2 and C3 toxins, as well as toxins from C.perfringens, C. spiriforma and C. difficile. These toxins are composedof a monomeric, enzymatically active A subunit which is responsible forADP-ribosylation of GTP-binding proteins, and a non-toxic B subunitwhich binds receptors on the surface of the target cell and delivers theA subunit across the cell membrane.

In the case of CT and LT, the A subunit is known to increaseintracellular cAMP levels in target cells, while the B subunit ispentameric and binds to GM1 ganglioside receptors. (LT-B also bindsadditional receptors.)

Previously, observations were made demonstrating that CT is able toinduce mucosal and systemic immunity against itself when administeredintraduodenally (i.e. to a mucosal surface). The membrane-bindingfunction of CT was shown to be essential for mucosal immunogenicity, butcholera toxoid, also known as the B subunit of CT (CT-B) was inactive inisolation (Pierce and Gowans, J. Exp. Med. 1975; 142: 1550; Pierce, J.Exp Med. 1978; 148: 195-206).

Subsequently, it was demonstrated that native CT induced immunity toco-administered antigens (Elson, Curr. Top. Microbiol. Immunol., 1989;146:29; Elson and Ealding, J. Immunol. 1984; 133:2892-2897; Elson andEalding, Ibid. 1984; 132:2736-2741; Elson et al., J. Immunol. Meth.1984; 67:101-118; Lycke and Homgren, Immunology 1986; 59:301-339) andthat immune responses may be elicted to diptheria or tetanus toxoidswhen these antigens are applied to skin in combination with CT.

Two approaches have been taken in order to address the problem of CTtoxicity. The first approach has involved the use of CT-B as a mucosaladjuvant. CT-B is entirely non-toxic. An adjuvant effect forco-administered CT-B has been alleged in a number of publications(Tamura et al., J. Immunol. 1992; 149:981-988; Hirabayashi et al.,Immunology 1992; 75: 493-498; Gizurarson et al., Vaccine 1991;9:825-832; Kikuta et al., Vaccine 1970; 8:595-599; Hirabayashi et al.Ibid. 1990; 8:243-248; Tamura et al., Ibid. 1989; 7:314-32-; Tamura etal., Ibid. 1989; 7:257-262; Tamura et al., Ibid. 1988; 6:409-413;Hirabayashi et al., Immunology 1991; 72:329-335 Tamura et al., Vaccine1989; 7:503-505).

However, a number of aspects of the observations reported above were notentirely convincing. For example, it was noted that the adjuvant effectascribed to CT-B was not H-2 (MHC) restricted. It is known, however,that the immune response to CTB is H-2 (MHC) restricted (Elson andEalding, Eur. J. Immuno. 1987; 17:425-428). Moreover, the allegedadjuvant effect was observed even in individuals already immune to CT-B.

Other groups were unable to observe any mucosal adjuvant effectattributable to CT-B (Lycke and Holmgren, Immunology 1986; 59:301-308;Lycke, et al., Eur. J. Immunol. 1992: 22:2277-2281). Experiments withrecombinant CT-B (Holmgren et al., Vaccine 1993; 11:1179-1183) confirmedthat the alleged effect is largely, if not exclusively, attributable tolow levels of contamination of CT-B preparations with CT.

A second approach to eliminating the toxicity of CT has been to mutatethe active holotoxin in order to reduce or eliminate its toxicity. Thetoxicity of CT resides in the A subunit and a number of mutants to CTand its homologue, LT, comprising point mutations in the A subunit, areknown in the art. See, for example, International Patent ApplicationWO92/19265. It is accepted in the art that CT and LT are generallyinterchangeable, showing considerable homology. ADP-ribosylatingbacterial toxin mutants have been shown to act as mucosal adjuvants, seeInternational Patent Application WO95/17211.

SUMMARY OF THE INVENTION

Accordingly, there remains a need for an active adjuvant which may beused to increase the immunogenicity of an antigen when administeredparenterally, such as intramuscularly, subcutaneously, intravenously,transcutaneously or intradermally. The present invention provides forsuch parenteral adjuvants in the form of non-toxic ADP ribosylatingbacterial toxins. It is demonstrated herein that such mutants, lackingtoxicity, are active as parenteral adjuvants and produce high antibodytiters and/or induction of cytotoxic T-lymphocytes (CTLs).

In one embodiment, then, the subject invention is directed to aparenteral adjuvant composition comprising a detoxified mutant of abacterial ADP-ribosylating toxin as the parenteral adjuvant and at leastone selected antigen.

In another embodiment, the invention is directed to a parenteraladjuvant composition comprising a detoxified mutant of a bacterialADP-ribosylating toxin as the parenteral adjuvant and a pharmaceuticallyacceptable topical vehicle.

In yet another embodiment, the invention is directed to a parenteraladjuvant composition comprising a detoxified mutant of a bacterialADP-ribosylating toxin as the parenteral adjuvant, a pharmaceuticallyacceptable topical vehicle and at least one selected antigen.

In another embodiment, the invention is directed to a method for makinga parenteral adjuvant composition comprising combining a detoxifiedmutant of a bacterial ADP-ribosylating toxin as the parenteral adjuvantwith at least one selected antigen.

In still a further embodiment, the invention is directed to a method ofmaking a parenteral adjuvant composition comprising combining adetoxified mutant of a bacterial ADP-ribosylating toxin as theparenteral adjuvant with a pharmaceutically acceptable topical vehicle.

In another embodiment, the invention is directed to a method forimmunizing a vertebrate subject comprising parenterally administering tothe vertebrate subject an immunologically effective amount of

a) an adjuvant comprising a detoxified mutant of a bacterialADP-ribosylating toxin in combination with a pharmaceutically acceptablevehicle; and

b) at least one selected antigen.

In particularly preferred embodiments, the non-toxic adjuvant is adetoxified mutant selected from the group consisting of cholera toxin(CT), pertussis toxin (PT), and an E. coli heat-labile toxin (LT),particularly LT-K63, LT-R72, CT-S109, and PT-K9/G129.

These and other embodiments of the present invention will readily occurto those of ordinary skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B show the DNA and corresponding amino acid sequences of awild-type subunit A from an E. coli heat labile toxin (LT) (SEQ ID NOS:1and 2) and a cholera toxin (CT) (SEQ ID NOS:3 and 4).

FIG. 2 shows the serum anti-LT antibody response followingtranscutaneous administration of representative adjuvant compositions ofthe present invention. Circles indicate titers from individual mice. Ifless than five circles are visible per group, two or more values wereidentical and circles were superimposed. Full triangles indicate meantiters per group±standard deviation.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See e.g.,Sambrook, et al., MOLECULAR CLONING; A LABORATORY MANUAL, SECOND EDITION(1989); DNA CLONING, VOLUMES I AND II (D. N Glover ed. 1985);OLIGONUCLEOTIDE SYNTHESIS (M. J. Gait ed, 1984); NUCLEIC ACIDHYBRIDIZATION (B. D. Hames & S. J. Higgins eds. 1984); TRANSCRIPTION ANDTRANSLATION (B. D. Hames & S. J. Higgins eds. 1984); ANIMAL CELL CULTURE(R. I. Freshney ed. 1986); IMMOBILIZED CELLS AND ENZYMES (IRL Press,1986); B. Perbal, A PRACTICAL GUIDE TO MOLECULAR CLONING (1984); theseries, METHODS IN ENZYMOLOGY (Academic Press, Inc.); GENE TRANSFERVECTORS FOR MAMMALIAN CELLS (J. H. Miller and M. P. Calos eds. 1987,Cold Spring Harbor Laboratory), METHODS IN ENZYMOLOGY Vol. 154 and Vol.155 (Wu and Grossman, and Wu, eds., respectively), Mayer and Walker,eds. (1987), IMMUNOCHEMICAL METHODS IN CELL AND MOLECULAR BIOLOGY(Academic Press, London), Scopes, (1987), PROTEIN PURIFICATION:PRINCIPLES AND PRACTICE, Second Edition (Springer-Verlag, New York), andHANDBOOK OF EXPERIMENTAL IMMUNOLOGY, VOLUMES I-IV (D. M. Weir and C. C.Blackwell eds 1986).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise.

The following amino acid abbreviations are used throughout the text:Alanine (Ala) A Arginine (Arg) R Asparagine (Asn) N Aspartic acid (Asp)D Cysteine (Cys) C Glutatmine (Gln) Q Glutamic acid (Glu) E Glycine(Gly) G Histidine (His) H Isoleucine (Ile) I Leucine (Leu) L Lysine(Lys) K Methionine Met) M Phenylalanine (Phe) F Proline (Pro) P Serine(Ser) S Threonine (Thr) T Tryptophan (Trp) W Tyrosine (Tyr) Y Valine(Val) V

I. Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

By “parenteral” is meant introduction into the body outside of thedigestive tract, such as by subcutaneous, intramuscular, transcutaneous,intradermal, or intravenous administration. This is to be contrastedwith adjuvants that are delivered to a mucosal surface, such as oral,intranasal, vaginal, or rectal.

As used herein, “detoxified” refers to both completely nontoxic and lowresidual toxic mutants of the toxin in question. Preferably, thedetoxified protein retains a toxicity of less than 0.01% of thenaturally occurring toxin counterpart, more preferably less than 0.001%and even more preferable, less than 0.0001% of the toxicity of thenaturally occurring toxin counterpart. The toxicity may be measured inmouse CHO cells or preferably by evaluation of the morphological changesinduced in Y1 cells. In particular, Y1 cells are adrenal tumorepithelial cells which become markedly more rounded when treated with asolution containing CT or LT (Ysamure et al., Cancer Res. (1966)26:529-535). The toxicity of CT and LT is correlated with thismorphological transition. Thus, the mutant toxins may be incubated withY1 cells and the morphological changes of the cells assessed.

The term “toxoid” as used herein means a genetically detoxified toxin.

By “antigen” is meant a molecule which contains one or more epitopes(either linear, conformational or both) that will stimulate a host'simmune system to make a cellular antigen-specific immune response whenthe antigen is produced, or a humoral antibody response. Such epitopesmay include from about 3 to about 20 or more amino acids. Normally, a Bcell epitope will include at least about 5 amino acids but can be assmall as 3-4 amino acids. A T cell epitope, such as a CTL epitope, willinclude at least about 7-9 amino acids, and a helper T cell epitope atleast about 12-20 amino acids. The term denotes both subunit antigens,i.e., antigens which are separate and discrete from a whole organismwith which the antigen is associated in nature, as well as killed,attenuated or inactivated bacteria, viruses, parasites or othermicrobes. Antibodies such as anti-idiotype antibodies, or fragmentsthereof, and synthetic peptide mimotopes, which can mimic an antigen orantigenic determinant, are also captured under the definition of antigenas used herein.

For purposes of the present invention, antigens can be derived from anyof several known viruses, bacteria, parasites and fungi. The term alsointends any of the various tumor antigens. Furthermore, for purposes ofthe present invention, an “antigen” refers to a protein which includesmodifications, such as deletions, additions and substitutions (generallyconservative in nature), to the native sequence, so long as the proteinmaintains the ability to elicit an immunological response. Thesemodifications may be deliberate, as through site-directed mutagenesis,or may be accidental, such as through mutations of hosts which producethe antigens.

An “immunological response” to an antigen or composition is thedevelopment in a subject of a humoral and/or a cellular immune responseto molecules present in the composition of interest. For purposes of thepresent invention, a “humoral immune response” refers to an immuneresponse mediated by antibody molecules, while a “cellular immuneresponse” is one mediated by T-lymphocytes and/or other white bloodcells. T cells can be divided into two major groups called CD8⁺ T orCD4⁺ T cells based on expression of either the CD8 or CD4 protein ontheir surface. CD8⁺ T cells are often referred to as cytotoxic T cells(CTL) and CD4⁺ T cells are often called helper T cells (Th). The Thcells can be further divided into Th1 and Th2 cells. In contrast to theB cells, T cells are not capable of recognizing native antigen butrequire specific processing of such antigens. Fragments of the antigenare presented by antigen presenting cells (APCs) to T cells. Thesefragments are associated with a specific protein on the surface of theAPC. CD8⁺ T cells recognize the fragment presented by MHC I proteinwhereas CD4⁺ T cells recognize antigenic fragments presented by MHC IIproteins.

One important aspect of cellular immunity involves an antigen-specificresponse by CTLs. CTLs have specificity for peptide antigens that arepresented in association with proteins encoded by the majorhistocompatibility complex (MHC) and expressed on the surfaces of cells.CTLs help induce and promote the intracellular destruction ofintracellular microbes, or the lysis of cells infected with suchmicrobes. Another aspect of cellular immunity involves anantigen-specific response by helper T cells. Helper T cells act to helpstimulate the function, and focus the activity of, nonspecific effectorcells against cells displaying peptide antigens in association with MHCmolecules on their surface. A “cellular immune response” also refers tothe production of cytokines, chemokines and other such moleculesproduced by activated T cells and/or other white blood cells, includingthose derived from CD4+ and CD8+ T cells.

A composition or vaccine that elicits a cellular immune response mayserve to sensitize a vertebrate subject by the presentation of antigenin association with MHC molecules at the cell surface. The cell-mediatedimmune response is directed at, or near, cells presenting antigen attheir surface. In addition, antigen-specific T-lymphocytes can begenerated to allow for the future protection of an immunized host.

The ability of a particular antigen to stimulate a cell-mediatedimmunological response may be determined by a number of assays, such asby lymphoproliferation (lymphocyte activation) assays, CTL cytotoxiccell assays, or by assaying for T-lymphocytes specific for the antigenin a sensitized subject. Such assays are well known in the art. See,e.g., Erickson et al., J. Immunol. (1993) 151:4189-4199; Doe et al.,Eur. J. Immunol. (1994) 24:2369-2376; and the examples below.

Thus, an immunological response as used herein may be one whichstimulates the production of CTLs, and/or the production or activationof helper T cells. The antigen of interest may also elicit anantibody-mediated immune response. Hence, an immunological response mayinclude one or more of the following effects: the production ofantibodies by B-cells; and/or the activation of suppressor T-cellsand/or γδ T-cells directed specifically to an antigen or antigenspresent in the composition or vaccine of interest. These responses mayserve to neutralize infectivity, and/or mediate antibody-complement, orantibody dependent cell cytotoxicity (ADCC) to provide protection to animmunized host. Such responses can be determined using standardimmunoassays and neutralization assays, well known in the art. For ageneral overview of the immune system and immunological mechanisms seefor example: Janeway, C. A. and Travers, P., IMMUNOBIOLOGY, 2nd ed.1996, Current Biology Ltd./Garland Publishing, New York, N.Y.

A composition which contains a selected antigen along with a detoxifiedmutant of a bacterial ADP-ribosylating toxin of the present invention,or a vaccine composition which is coadministered with the subjectadjuvant, displays “enhanced immunogenicity” when it possesses a greatercapacity to elicit an immune response than the immune response elicitedby an equivalent amount of the antigen administered without theadjuvant. Thus, a vaccine composition may display “enhancedimmunogenicity” because the antigen is more strongly immunogenic orbecause a lower dose or fewer doses of antigen are necessary to achievean immune response in the subject to which the antigen is administered.Such enhanced immunogenicity can be determined by administering theadjuvant composition and antigen controls to animals and comparingantibody titers and/or cellular-mediated immunity against the two usingstandard assays such as radioimmunoassay, ELISAs, CTL assays, and thelike, well known in the art.

For purposes of the present invention, an “effective amount” of anadjuvant will be that amount which enhances an immunological response toa coadministered antigen.

As used herein, “treatment” refers to any of (i) prevention of infectionor reinfection, as in a traditional vaccine, (ii) reduction orelimination of symptoms, and (iii) reduction or complete elimination ofthe pathogen in question. Treatment may be effected prophylactically(prior to infection) or therapeutically (following infection).

By “vertebrate subject” is meant any member of the subphylum cordata,including, without limitation, humans and other primates, includingnon-human primates is such as chimpanzees and other apes and monkeyspecies; farm animals such as cattle, sheep, pigs, goats and horses;domestic mammals such as dogs and cats; laboratory animals includingrodents such as mice, rats and guinea pigs; birds, including domestic,wild and game birds such as chickens, turkeys and other gallinaceousbirds, ducks, geese, and the like; and fish. The term does not denote aparticular age. Thus, both adult and newborn individuals are intended tobe covered. The system described above is intended for use in any of theabove vertebrate species, since the immune systems of all of thesevertebrates operate similarly.

II. Modes of Carrying Out the Invention

The present invention is based on the surprising and unexpecteddiscovery that detoxified mutants of bacterial ADP-ribosylating toxins,such as CT, LT and PT, are able to serve as parenteral adjuvants toenhance humoral and/or cell-mediated immune responses in a vertebratesubject when the adjuvants are administered with a selected antigen.Since the present adjuvants function when administered parenterally,they permit a convenient method of conferring immunity to substancesthat are not amenable to other modes of administration. Accordingly, thepresent system is useful with a wide variety of antigens and provides apowerful tool to prevent and/or treat a large number of infections.

Regarding the present invention, any detoxified mutant of a bacterialADP-ribosylating toxin can be used as a parenteral adjuvant. Suchmutants optionally comprise one or more amino acid additions, deletionsor substitutions that result in a molecule having reduced toxicity whileretaining adjuvanticity. If an amino acid is substituted for thewild-type amino acid, such substitutions may be with a naturallyoccurring amino acid or may be with a modified or synthetic amino acid.Substitutions which alter the amphotericity and hydrophilicity whileretaining the steric effect of the substituting amino acid as far aspossible are generally preferred.

The mutants of the invention are preferably in the form of a holotoxin,comprising the mutated A subunit and the B subunit, which may beoligomeric, as in the wild-type holotoxin. The B subunit is preferablynot mutated. However, it is envisaged that a mutated A subunit may beused in isolation from the B subunit, either in an essentially pure formor complexed with other agents, which may replace the B subunit and/orits functional contribution.

As explained above, in addition to the completely nontoxicADP-ribosylating bacterial toxins, toxins can be used wherein a residualtoxicity greater than 100 to 10,000 fold lower, or more, than itsnaturally occurring counterparts is found.

Particularly suitable are detoxified mutants of diphtheria toxin, CT,LT, or PT; such mutations are known in the art. For example, particularmutant LTs in accordance with the invention may possess the followingmutations of the A subunit: a Val to Asp, Glu or Tyr substitution atposition 53; a Ser to Lys substitution at position 63 (termed LT-K63herein); an Ala to Arg substitution at position 72 (termed LT-R72herein); a Val to Lys or Tyr substitution at position 97; a Tyr to Lys,Asp or Ser substitution at position 104; a Pro to Ser substitution atposition 106; an Arg to Gly substitution at position 192.

Since the amino acid sequences of CT-A and LT-A are substantiallyconserved (see FIGS. 1A-1B, (SEQ ID NOS:1-4)), the changes describedabove with respect to LT can also be made to the corresponding positionsin CT. A particularly preferred CT mutant comprises a substitution ofSer at position 109 (termed CT-S109 herein).

A preferred detoxified mutant of Bordetella pertussis is a double mutantwhere Lys replaces Arg at amino acid position 9 and Gly replaces Glu atamino acid position 129 (termed PT-K9/G129 herein). Many other suitablepertussis toxin (PT) mutants are known in the art.

Methods for the design and production of mutants of CT and/or LT areknown in the art. Suitable methods are described in International Patentapplication WO93/13202, as well as WO92/19265. In particular, suchmutant toxins may be synthesized chemically, using conventional peptidesynthesis techniques. See, e.g., See, e.g., J. M. Stewart and J. D.Young, Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co.,Rockford, Ill. (1984) and G. Barany and R. B. Merrifield, The Peptides:Analysis, Synthesis, Biology, editors E. Gross and J. Meienhofer, Vol.2, Academic Press, New York, (1980), pp. 3-254, for solid phase peptidesynthesis techniques; and M. Bodansky, Principles of Peptide Synthesis,Springer-Verlag, Berlin (1984) and E. Gross and J. Meienhofer, Eds., ThePeptides: Analysis, Synthesis, Biology, supra, Vol. 1, for classicalsolution synthesis.

Alternatively, and preferably, mutations can be made to the wild-typesequence using conventional recombinant techniques such as by preparingsynthetic oligonucleotides including the mutations and inserting themutated sequence into the gene encoding the wild-type protein usingrestriction endonuclease digestion. (See, e.g., Kunkel, T. A. Proc.Natl. Acad. Sci. USA (1985) 82:448; Geisselsoder et al. BioTechniques(1987) 5:786.) Alternatively, the mutations can be effected using amismatched primer which hybridizes to the wild-type nucleotide sequence(generally cDNA corresponding to the RNA sequence), at a temperaturebelow the melting temperature of the mismatched duplex. The primer canbe made specific by keeping primer length and base composition withinrelatively narrow limits and by keeping the mutant base centrallylocated. Zoller and Smith, Methods Enzymol. (1983) 100:468. Primerextension is effected using DNA polymerase, the product cloned andclones containing the mutated DNA, derived by segregation of the primerextended strand, selected. Selection can be accomplished using themutant primer as a hybridization probe. The technique is also applicablefor generating multiple point mutations. See, e.g., Dalbie-McFarland etal. Proc. Natl. Acad. Sci USA (1982) 79:6409. PCR mutagenesis will alsofind use for effecting the desired mutations.

The adjuvant of the invention is preferably administered in admixturewith at least one antigen against which it is desired to raise an immuneresponse. If the antigen and the adjuvant are not in admixture, it ispreferred that they be administered within a relatively short time ofeach other, at the same site of administration, although there may be adelay of up to 5 days and a two-injection site regime. Thus, theadjuvant may be administered prior or subsequent to, or concurrent withthe selected antigen. It has been observed that the adjuvant effectprovided by wild-type CT is short-lived (see Lycke and Homgren,Immunology 1986: 59: 301-308).

In an alternative embodiment, the adjuvant of the present invention maybe administered, optionally in isolation from other antigens, as a boostfollowing systemic or mucosal administration of a vaccine.

Diseases against which the subject may be immunized include all diseasescapable of being treated or prevented by immunization, including viraldiseases, allergic manifestations, diseases caused by bacterial or otherpathogens, such as parasitic organisms, AIDS, autoimmune diseases suchas Systemic Lupus Erythematosus, Alzheimer's disease and cancers.Vaccine formulations suitable for delivery may be prepared as set outhereinbelow, while further formulations will be apparent to those ofstill in the art.

Thus, the antigen may be any antigen to which it is desired to raise animmune response in the subject. Suitable antigens comprise bacterial,viral, fungal and protozoan antigens derived from pathogenic organisms,as well as allergens, and antigens derived from tumors andself-antigens. Typically, the antigen will be a protein, polypeptide orpeptide antigen, but alternative antigenic structures, such as nucleicacid antigens, carbohydrate antigens and whole or attenuated orinactivated organisms such as bacteria, viruses or protozoa areincluded.

Specific examples of antigens useful in the present invention include awide variety of proteins from the herpesvirus family, including proteinsderived from herpes simplex virus (HSV) types 1 and 2, such as HSV-1 andHSV-2 glycoproteins gB, gD and gH; antigens derived from varicellazoster virus (VZV), Epstein-Barr virus (EBV) and cytomegalovirus (CMV)including CMV gB and gH; and antigens derived from other humanherpesviruses such as HHV6 and HHV7. (See, e.g. Chee et al.,Cytomegaloviruses (J. K. McDougall, ed., Springer-Verlag 1990) pp.125-169, for a review of the protein coding content of cytomegalovirus;McGeoch et al., J. Gen. Virol. (1988) 69:1531-1574, for a discussion ofthe various HSV-1 encoded proteins; U.S. Pat. No. 5,171,568 for adiscussion of HSV-1 and HSV-2 gB and gD proteins and the genes encodingtherefor; Baer et al., Nature (1984) 310:207-211, for the identificationof protein coding sequences in an EBV genome; and Davison and Scott, J.Gen. Virol. (1986) 67:1759-1816, for a review of VZV.)

The adjuvant compositions of the present invention can also be used todeliver antigens from the hepatitis family of viruses, includinghepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus(HCV), the delta hepatitis virus (HDV), hepatitis E virus (HEV) andhepatitis G virus (HGV). By way of example, the viral sequence of HCV isknown, as are methods for obtaining the sequence. See, e.g.,International Publication Nos. WO 89/04669; WO 90/11089; and WO90/14436. The HCV genome encodes several viral proteins, including E1(also known as E) and E2 (also known as E2/NSI). (See, Houghton et al.,Hepatology (1991) 14:381-388, for a discussion of HCV proteins,including E1 and E2.) The sequences of these proteins, as well asantigenic fragments thereof, will find use in the present methods.Similarly, the sequence for the δ-antigen from HDV is known (see, e.g.,U.S. Pat. No. 5,378,814) and this antigen can also be conveniently usedin the present methods.

Antigens derived from other viruses will also find use in the claimedmethods, such as without limitation, proteins from members of thefamilies Picornaviridae (e.g., polioviruses, etc.); Caliciviridae;Togaviridae (e.g., rubella virus, dengue virus, etc.); Flaviviridae;Coronaviridae; Reoviridae; Birnaviridae; Rhabodoviridae (e.g., rabiesvirus, etc.); Filoviridae; Paramyxoviridae (e.g., mumps virus, measlesvirus, respiratory syncytial virus, etc.); Orthomyxoviridae (e.g.,influenza virus types A, B and C, etc.); Bunyaviridae; Arenaviridae;Retroviradae (e.g., HTLV-I; HTLV-II; HIV-1 (also known as HTLV-III, LAV,ARV, hTLR, etc.)), including but not limited to antigens from theisolates HIV_(IIIb), HIV_(SF2) , HIV_(LAV), HIV_(LAI), HIV_(MN));HIV-1_(CM235), HIV-1_(US4); HIV-2; simian immundeficiency virus (SIV)among others. See, e.g. Virology, 3rd Edition (W. K. Joklik ed. 1988);Fundamental Virology, 2nd Edition (B. N. Fields and D. M. Knipe, eds.1991), for a description of these and other viruses.

For example, the present adjuvants can be used in conjunction with thegp120 envelope protein from HIV_(SF2), HIV-1_(CM235), HIV-1_(US4),HIV-1_(IIIB) and HIV-1_(LAI). The gp120 sequences for these and amultitude of additional HIV-1 and HIV-2 isolates, including members ofthe various genetic subtypes of HIV, are known and reported (see, e.g.,Myers et al., Los Alamos Database, Los Alamos National Laboratory, LosAlamos, N. Mex. (1992); Myers et al., Human Retroviruses and Aids, 1990,Los Alamos, N. Mex.: Los Alamos National Laboratory; and Modrow et al.,J. Virol. (1987) 61:570-578, for a comparison of the envelope sequencesof a variety of HIV isolates) and sequences derived from any of theseisolates will find use in the present methods. Furthermore, theinvention is equally applicable to other immunogenic proteins derivedfrom any of the various HIV isolates, including any of the variousenvelope proteins such as gp160 and gp41, gag antigens such as p24gagand p55gag, as well as proteins derived from the pol region.

Additionally, the envelope glycoproteins HA and NA of influenza A are ofparticular interest for generating an immune response. Numerous HAsubtypes of influenza A have been identified (Kawaoka et al., Virology(1990) 179:759-767; Webster et al., “Antigenic variation among type Ainfluenza viruses,” p. 127-168. In: P. Palese and D. W. Kingsbury (ed.),Genetics of influenza viruses. Springer-Verlag, New York). Thus,proteins derived from any of these isolates can also be used in thetechniques described herein.

The compositions and methods described herein will also find use withnumerous bacterial antigens, such as those derived from organisms thatcause diphtheria, cholera, tuberculosis, tetanus, pertussis, meningitis,and other pathogenic states, including, without limitation, Bordetellapertussis, Neisseria meningitides (A, B, C, Y), Hemophilus influenzatype B (HIB), and Helicobacter pylori. Examples of parasitic antigensinclude those derived from organisms causing malaria and Lyme disease.

Furthermore, the methods and compositions described herein provide ameans for treating a variety of malignant cancers. For example, thesystem of the present invention can be used to mount both humoral andcell-mediated immune responses to particular proteins specific to thecancer in question, such as an activated oncogene, a fetal antigen, oran activation marker. Such tumor antigens include any of the variousMAGEs (melanoma associated antigen E), including MAGE 1, 2, 3, 4, etc.(Boon, T. Scientific American (March 1993) :82-89); any of the varioustyrosinases; MART 1 (melanoma antigen recognized by T cells), mutantras; mutant p53; p97 melanoma antigen; CEA (carcinoembryonic antigen),among others.

It is readily apparent that the subject invention can be usedprophylactically (to prevent disease) or therapeutically (to treatdisease after infection) for a wide variety of diseases. Not only arethe compositions herein useful for preventing or treating disease, thesubject compositions may also be used to prepare antibodies, bothpolyclonal and monoclonal, useful for, e.g., diagnostic purposes, aswell as for immunopurification of particular antigens against which theyare directed.

If polyclonal antibodies are desired, a selected mammal, (e.g., mouse,rabbit, goat, horse, etc.) is immunized with the adjuvant compositionsof the present invention, along with the desired antigen. In order toenhance immunogenicity, the antigen can be linked to a carrier prior toimmunization. Immunization for the production of antibodies is generallyperformed by injecting the composition parenterally (generallysub-cutaneously or intramuscularly). The animal is usually boosted 2-6weeks later with one or more injections of the antigen, with theadjuvant compositions described herein or with alternate adjuvants.Antibodies may also be generated by in vitro immunization, using methodsknown in the art. Polyclonal antisera is then obtained from theimmunized animal and treated according to known procedures. See, e.g.,Jurgens et al. (1985) J. Chrom. 348:363-370.

Monoclonal antibodies are generally prepared using the method of Kohlerand Milstein, Nature (1975) 256:495-96, or a modification thereof.Typically, a mouse or rat is immunized as described above. However,rather than bleeding the animal to extract serum, the spleen (andoptionally several large lymph nodes) is removed and dissociated intosingle cells. If desired, the spleen cells may be screened (afterremoval of non-specifically adherent cells) by applying a cellsuspension to a plate or well coated with the protein antigen. B cells,expressing membrane-bound immunoglobulin specific for the antigen, willbind to the plate, and are not rinsed away with the rest of thesuspension. Resulting B cells, or all dissociated spleen cells, are theninduced to fuse with myeloma cells to form hybridomas, and are culturedin a selective medium (e.g., hypoxanthine, aminopterin, thymidinemedium, “HAT”). The resulting hybridomas are plated by limitingdilution, and are assayed for the production of antibodies which bindspecifically to the immunizing antigen (and which do not bind tounrelated antigens). The selected monoclonal antibody-secretinghybridomas are then cultured either in vitro (e.g., in tissue culturebottles or hollow fiber reactors), or in vivo (as ascites in mice) .See, e.g., M. Schreier et al., Hybridoma Techniques (1980); Hammerlinget al., Monoclonal Antibodies and T-cell Hybridomas (1981); Kennett etal., Monoclonal Antibodies (1980); see also U.S. Pat. Nos. 4,341,761;4,399,121; 4,427,783; 4,444,887; 4,452,570; 4,466,917; 4,472,500,4,491,632; and 4,493,890. Panels of monoclonal antibodies producedagainst the hormone of interest, or fragment thereof, can be screenedfor various properties; i.e., for isotype, epitope, affinity, etc.

Compositions according to the invention may comprise one or moreantigens. Furthermore, one or more “pharmaceutically acceptableexcipients or vehicles” are present, such as water, saline, glycerol,polyethyleneglycol, hyaluronic acid, ethanol, etc. Additionally,auxiliary substances, such as wetting or emulsifying agents, pHbuffering substances, and the like, may be present in such vehicles.

A carrier is optionally present that does not itself induce theproduction of antibodies harmful to the individual receiving thecomposition. Suitable carriers are typically large, slowly metabolizedmacromolecules such as proteins, polysaccharides, polylactic acids,polyglycolic acids, polymeric amino acids, amino acid copolymers, lipidaggregates (such as oil droplets or liposomes), and inactive virusparticles. Such carriers are well known to those of ordinary skill inthe art. Additionally, these carriers may function as furtherimmunostimulating agents (“adjuvants”).

Typically, the compositions are prepared as injectables, either asliquid solutions or suspensions; solid forms suitable for solution in,or suspension in, liquid vehicles prior to injection may also beprepared. The preparation may also be emulsified or the activeingredient encapsulated in liposome vehicles. Furthermore, compositionssuitable for topical use may also be formulated. For example, theadjuvant compositions may be provided in the form of pharmaceuticallyacceptable topical vehicles such as ointments, creams, gels andemulsions. Ointments, creams and emulsions containing the adjuvants canbe prepared using known techniques. A variety of suitable pharmaceuticalointment bases are generally known, including oleaginous bases,anhydrous absorption bases, and oil-in-water (o/w) bases. Oleaginousbases include petrolatum or petrolatum modified by waxes (e.g., liquidpetrolatum gelled by the addition of a polyethylene) and those preparedfrom vegetable fixed oils or animal fats (e.g., lard, benzoinated lard,olive oil, cottonseed oil, or the like). Anhydrous bases includehydrophilic petrolatum, anhydrous lanolin and lanolin derivatives.Oil-in-water bases (e.g., emulsion bases or creams) generally includethree parts, the oil phase, the emulsifier and the aqueous phase. Theadjuvant, and optionally the antigen, can be included in any one of thephases, or added to the formed emulsion. The oil phase is typicallycomprised of petrolatum with one or more higher molecular weightalcohols such as cetyl or steryl alcohol. The aqueous phase generallycontains preservatives, the water-soluble components of the emulsionsystem, humectants (such as glycerin, propylene glycol or a polyethyleneglycol), as well as optional stabilizers, antioxidants, buffers and thelike.

The above pharmaceutical ointments are formed by dispersing finelydivided or dissolved adjuvant and, optionally one or more selectedantigens, uniformly throughout the vehicle or base. Creams, lotions andemulsions can be formed by way of a two-phase heat system, whereinoil-phase ingredients are combined under heat to provide a liquified,uniform system. The aqueous-phase ingredients are separately combinedusing heat. The oil and aqueous phases are then added together withconstant agitation and allowed to cool. At this point, concentratedagents may be added as a slurry. Volatile or aromatic materials can beadded after the emulsion has sufficiently cooled. Preparation of suchpharmaceutical compositions is within the general skill of the art. See,e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company,Easton, Pa., 18th edition, 1990.

The adjuvants can also be incorporated into gel formulations using atwo-phase gel system. Such systems generally comprise a suspension ornetwork of small, discrete particles interpenetrated by a liquid toprovide a dispersed phase and a liquid phase. Single-phase gel systemsare formed by distributing organic macromolecules uniformly throughout aliquid such that there are no apparent boundaries between the dispersedand liquid phases. Suitable gelling agents for use herein includesynthetic macromolecules (e.g., Carbomers®, polyvinyl alcohols andpolyoxyethylene-polyoxypropylene copolymers), gums such as tragacanth,as well as sodium alginate, gelatin, methylcellulose, sodiumcarboxymethylcellulose, methylhydroxyethyl cellulose and hydroxyethylcellulose. In order to prepare a uniform gel, dispersing agents such asalcohol or glycerin can be added, or the gelling agent can be dispersedby trituration, mechanical mixing or stirring, or combinations thereof.

Lotion preparations are generally liquid or semiliquid preparationscontaining the adjuvant and, optionally, one or more selected antigens,in a suitable vehicle. Lotions are formed by suspending finely dividedsolids in an aqueous medium. Optional dispersing agents can be employedto aid in the preparation of the liquid formulation, as well as one ormore surface-active agents.

In the cream and ointment formulations described hereinabove, optionalingredients can include materials such as antioxidants, viscositymodifiers (e.g., paraffin wax or lanolin wax), and topical absorptionrate modifiers. Actual methods of preparing any of the aboveformulations are known, or will be apparent, to those skilled in theart. See, e.g., Remington's Pharmaceutical Sciences, Mack PublishingCompany, Easton, Pa., 18th edition, 1990.

Immunogenic compositions used as vaccines comprise an immunologicallyeffective amount of the adjuvant and an antigen, as well as any otherthe above-mentioned components, as needed. By “immunologically effectiveamount”, is meant that the administration of that amount to anindividual, either in a single dose or as part of a series, is such thatan immune response can be generated in the subject to which it isadministered. The exact amount necessary will vary depending on thesubject being treated; the age and general condition of the subject tobe treated; the capacity of the subject's immune system to synthesizeantibodies and/or mount a cell-mediated immune response; the degree ofprotection desired; the severity of the condition being treated; theparticular antigen selected and its mode of administration, among otherfactors. An appropriate effective amount can be readily determined byone of skill in the art. Thus, an “immunologically effective amount”will fall in a relatively broad range that can be determined throughroutine trials. In general, an “immunologically effective” amount ofantigen will be an amount on the order of about 0.1 μg to about 1000 μg,more preferably about 1 μg to about 100 μg.

Similarly, the adjuvant will be present in an amount such that theantigen displays “enhanced immunogenicity,” as defined above, ascompared to administration of the antigen alone, without the adjuvant.Amounts which are effective for eliciting an enhanced immune responsecan be readily determined by one of skill in the art.

Dosage treatment may be a single dose schedule or a multiple doseschedule. The vaccine may be administered in conjunction with otherimmunoregulatory agents.

Additional adjuvants can be used to enhance effectiveness; suchadjuvants include, but are not limited to: (1) aluminum salts (alum),such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc;(2) oil-in-water emulsion formulations (with or without other specificimmunostimulating agents such as muramyl peptides (see below) orbacterial cell wall components), such as for example (a) MF59 (PCT Publ.No. WO 90/14837), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span85 (optionally containing various amounts of MTP-PE (see below),although not required) formulated into submicron particles using amicrofluidizer such as Model 110Y microfluidizer (Microfluidics, Newton,Kans.), (b) SAF, containing 10%-Squalane, 0.4% Tween 80, 5%pluronic-blocked polymer L121, and thr-MDP (see below) eithermicrofluidized into a submicron emulsion or vortexed to generate alarger particle size emulsion, and (c) Ribi™ adjuvant system (RAS),(Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% Tween80, and one or more bacterial cell wall components from the groupconsisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM),and cell wall skeleton (CWS), preferably MPL+CWS (Detox™); (3) saponinadjuvants, such as Stimulon™ (Cambridge Rioscience, Worcester, Mass.)may be used or particles generated therefrom such as ISCOMs(immunostimulating complexes); (4) Complete Freunds Adjuvant (CFA) andIncomplete Freunds Adjuvant (IFA); (5) cytokines, such as interleukins(e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons(e.g., gamma interferon), macrophage colony stimulating factor (M-CSF),tumor necrosis factor (TNF), etc; and (6) other substances that act asimmunostimulating agents to enhance the effectiveness of thecomposition. Alum and MF59 are preferred. Muramyl peptides include, butare not limited to, N-acetyl-muramyl-L-threonyl-Disoglutamine (thr-MDP),N-acetyl-normuramyl-^(L)-alanyl-^(D)-isoglutamine (nor-MDP) ,N-acetylmuramyl-^(L)-alanyl-^(D)-isoglutaminyl-^(L)-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3 -huydroxyphosphoryloxy)-ethylamine(MTP-PE), etc.

The invention further provides a method for the manufacture of anadjuvanted vaccine comprising the steps of:

a) performing site-directed mutagenesis on the A subunit of a bacterialADP-ribosylating toxin in order to detoxify the toxin; and

b) combining the detoxified toxin with an antigen, such that itfunctions as a parenteral adjuvant.

III. Experimental

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

EXAMPLE 1 Production of LT-K63

LT-K63, for use in the following experiments, was made as follows.Site-directed mutagenesis was used to replace the Ser reside at position63 of the A subunit of LT with Lys in order to construct a non-toxic LTmutant that would still assemble as a holotoxin with cell bindingactivity. The mutant protein, named LT-K63, was purified and tested forADP-ribosyltransferase and toxic activity in several assays. LT-K63 wasstill able to bind GM1 ganglioside receptor but showed a complete lossof enzymatic activity, in agreement with published data (Lobet et al.,Infect. Immun. 1991; 59:2870-2879). Further, LT-K63 was inactive in themouse ileal loop assay and in vitro on Y1 cells.

EXAMPLE 2 Parenteral Adjuvant Activity of LT-K63 with HSV qD2 in Mice

The LT-K63 mutant, produced as described in Example 1, was tested as aparenteral adjuvant with herpes simplex virus type 2 (HSV-2) gD antigenas follows.

a. Mice were immunized twice by intramuscular injection one month apartwith a 10 μg dose of the LT-K63 mutant and a 10 μg dose of HSV-2 gD2antigen. Sera were collected on day 0 and two weeks after the secondimmunization (day 42). The antibody responses against HSV-2 gD2 weremeasured by ELISA. The geometric mean titers plus or minus standarderror are listed in Table 1. This experiment illustrates the ability ofthe LT-K63 mutant in combination with HSV-2 gD2 to produce an immuneresponse in mice. TABLE 1 Serum anti-gD2 titers of mice immunized withLT-K63 and gD2 Animal # Day 0 Day 42 BL898 <10 4510 BL899 <10 45920BL900 <10 7535 BL901 <10 56585 BL902 <10 74085 BL903 <10 8845 BL904 <104340 BL905 <10 19430 BL906 <10 6380 BL907 <10 9125 GMT +/− SEM 13975 +/−4726

b. Mice were immunized twice by intramuscular injection one month apartwith 10 μg HSV-2 gD2. Sera were collected on day 0 and two weeks afterthe second immunization. The antibody responses against HSV-2 gD2 weremeasured by ELISA and are shown as geometric mean titer plus or minusstandard error in Table 2. The 90-fold lower antibody response producedby HSV-2 gD2 (150) compared to HSV-2 gD2 combined with LT-K63 (13980)illustrates the parenteral adjuvant activity of LT-K63 with HSV-2 gD2.TABLE 2 Serum anti-HSV gD2 antibody titers of mice immunized with gD2Animal # Day 14 Sera Day 42 Sera BC047 <10 348 BC048 <10 11 BC049 <10 17BC050 <10 222 BC051 <10 73 BC052 <10 44 BC053 <10 2053 BC054 <10 2882GMT +/− SEM 151 +/− 111

EXAMPLE 3 Parenteral Adjuvant Activity of LT-K63 with Influenza HA inMice

Mice were immunized twice by intramuscular injection one month apartwith 1 μg LT-K63 (produced as described in Example 1) and 1 μg A/TexasHA (hemagglutinin) antigen or 1 pg A/Texas HA alone. Sera were collectedtwo weeks after the second immunization. The anti-HA ELISA titers areshown as geometric mean titer plus or minus standard error in Table 3.The 11-fold higher antibody response observed in the group receiving HAantigen combined with the LT-K63 mutant (70380) compared with the groupreceiving HA antigen alone (6390) illustrates the effectiveness ofLT-K63 as a parenteral adjuvant with influenza HA antigen. TABLE 3 Serumanti-HA titers of mice immunized with HA or HA with LT-K63 HA HA +LT-K63 Animal # Day 42 Animal # Day 42 CN 622 3698 CN 623 7778 CN 6245506 CN 625 5142 CN 626 7109 CN 627 7422 CN 628 51463 CN 629 19299 CN630 2906 CN 631 427 CN 632 2601 CN 642 73486 CN 633 4817 CN 643 70019 CN634 7315 CN 644 43773 CN 635 19056 CN 645 79454 CN 636 19979 CN 646229580 CN 637 2049 CN 647 43157 CN 638 3404 CN 648 29928 CN 639 12447 CN649 84437 CN 640 4817 CN 650 88956 CN 641 16752 CN 651 74790 GMT +/− SEM6391 +/− 1484 70378 +/− 12194

EXAMPLE 4 Parenteral Adjuvant Activity of LT-K63 with HIV p24 gag inMice

a. Mice were immunized three times by subcutaneous injection 1 weekapart with 10 μg LT-K63 (produced as described in Example 1) and 10 μgHIV p24 gag or 10 μg HIV p24 gag alone. HIV p24 gag-specific CTLactivity is depicted in Table 4. CTL activity was measured in a standardchromium release assay and is presented as % specific lysis. Inparticular, SVBalb (H-2d) and MC57 (H-2b) target cells were incubatedwith 51Cr and 1uM p7g peptide for 1 hour. Effector cells (E) werecultured with target cells (T) at various E:T ratios for 4 hours. Theaverage cpm from duplicate wells was used to calculate percent specific51Cr release. Allogeneic Mc57 target cells and syngeneic SvBalb targetcells had low background killing. Syngeneic SvBalb cells pulsed with HIVp24 gag epitope peptide p7g had 69% specific lysis at a 50:1 E:T ratiofor HIV p24 gag with LT-K63. In contrast, HIV p24 gag alone induced only29% killing under the same conditions. The group receiving LT-K63 had ahigher CTL response in contrast to the gag alone group. This illustratesthe adjuvant activity of LT-K63 for CTL induction with HIV p24 gag.TABLE 4 CTL Responses of Mice Immunized with HIV gag and LT-K63KEffector: % specific lysis Target Mc57/p7g SvB/p7g SvB/- LT-K63 + 50:1 269 7 HIV p24 10 3 31 7 gag  2 4 14 4 HIV p24 50:1 5 29 4 gag 10 3 12 2 2 2 5 3

b. Mice were immunized twice by subcutaneous injection one month apartwith 10 μg LT-K63 and 10 μg HIV p24 gag. Sera were collected two weeksafter the second immunization. The anti-HIV p24 gag titers are shown asgeometric mean titer plus or minus standard error in Table 5. Thisexperiment illustrates the ability of LT-K63 in combination with HIV p24gag to produce an anti-HIV p24 gag response in mice. TABLE 5 Antibodyresponses of mice immunized with p24 gag Animal # Day 0 Day 42 CP386 13220406 CP387 15 153674 CP388 9 235706 CP389 20 350167 GMT +/− SEM 229900+/− 38800

EXAMPLE 5 Transcutaneous Adjuvant Activity of LT-K63 and LT-R72

For the following experiments, the LT wild-type (LTwt), LT-K63 andLT-R72 mutants were obtained as described (Giuliani et al., “MucosalAdjuvanticity of LTR72, a Novel Mutant of Escherichia coli Heat-LabileEnterotoxin with Partial Knock-Out of ADP Ribosyltransferase Activity,”J. Exp. Med. 187:in press). The antigen used, CRM₁₉₇, is awell-characterized non-toxic diphtheria toxin mutant. See, e.g., Bixleret al. (1989) Adv. Exp. Med. Biol. 251:175, Constantino et al. (1992)Vaccine; International Publication No. WO 96/14086.

For transcutaneous immunization, on day 0, groups of 5 female BALB/cmice were anesthetized with an intraperitoneal injection of 100 μl/10 gof weight of a solution of Ketavet™ 50 (20% v/v), Rompun™ (3% v/v), andCombelen™ (3% v/v) in sterile saline. Mice were then shaved on the back(about 2 cm²), and 100 μl of phosphate-buffered saline (PBS) containing100 μg of CRM197 and 50 μg of LTwt or LT mutants were gently applied onthe shaved skin. Mice were kept under anesthesia for about 1 hour, thenwashed with lukewarm tap water, and dried. The same procedure wasrepeated on day 21. Third and fourth immunizations were performed on day51 and day 66, respectively. On the same dates, control groups of 5 micereceived CRM197 (10 μg) and LTwt (1 μg) intranasally (20 μl volume).

Serum samples were taken at days −1, 20, 35, 65, and 80. Antibodies toLT and CRM were determined by standard ELISA procedures.

No anti-CRM antibody response was detectable. As shown in Table 6 andFIG. 2, transcutaneous immunization induced a very strong anti-LTantibody response after one immunization (see Table 6), which wasboosted after the second immunization (see Table 6 and FIG. 2). Thus,transcutaneous immunization (i.e., application of soluble antigens plusmucosal adjuvants on the skin) induced the production of specificantibodies, showing that the immune system responded to the LT proteins.This result evidences that these proteins may be useful astranscutaneous adjuvants. TABLE 6 Serum anti-LT antibody titers inBALB/c mice immunized transcutaneously day −1 day day log (pre- 20 35titer mouse im- (post- (post- day n. mune) 1) 2) 35 mean SD Group 1 tc 1*0  60856 81127 4.91 5.34 0.37 LT w.t. 2 0 31319 833666 5.92 3 0 109229256225 5.41 4 0 129280 182907 5.26 5 0 35628 156077 5.19 Group 2 tc 6 00 0 CRM 7 0 0 0 8 0 0 0 9 0 0 0 10 0 0 0 Group 3 tc 11 0 9593 99577 5.004.98 0.31 CRM + LT 12 0 4606 73229 4.86 w.t. 13 0 3455 60058 4.78 14 05137 56589 4.75 15 0 20997 327216 5.51 Group 4 tc 16 0 7691 16501 4.223.90 0.49 CRM + 17 0 6307 37822 4.58 LTK63 18 0 404 2770 3.44 19 0 5725382 3.73 20 0 843 3278 3.52

TABLE 6 Serum anti-LT antibody titers in BALB/c mice immunizedtranscutaneously day −1 day day log (pre- 20 35 titer mouse im- (post-(post- day n. mune) 1) 2) 35 mean SD Group 5 tc 21 0 2401 25676 4.414.67 0.30 CRM + 22 0 6868 45181 4.65 LTR72 23 0 6868 33891 4.53 24 08049 38174 4.58 25 0 19452 186017 5.19 Group 6 26 0 169516 1195152 6.086.17 0.37 i.n. 27 0 104288 489685 5.69 CRM + 28 0 210832 4000000 6.60 LTw.t. 29 0 187184 989957 6.00 30 0 289546 3105000 6.49*0 = negative (titer < 50)

Groups 1 to 5 were immunized transcutaneously (tc), group 6intranasally. For tc immunizations (days 0 and 21), groups of BALB/cmice were shaved on the back (about 2 cm²) and kept inder anethesia for1 hour. During this time, 100 microliters of PBS containing antigenCRM197 (100 micrograms) and LT or LT mutants (50 micrograms) wereapplied on the shaved skin. Mice were then extensively washed withlukewarm water to avoid possible oral intake of residual antigens. Serumsamples were taken at the dates indicated, and tested by ELISA forquantitation of specific antibodies.

Thus, parenteral adjuvants comprising detoxified mutants of a bacterialADP-ribosylating toxin are disclosed. Although preferred embodiments ofthe subject invention have been described in some detail, it isunderstood that obvious variations can be made without departing fromthe spirit and the scope of the invention as defined by the appendedclaims.

1. A method for immunizing a vertebrate subject against at least oneselected antigen, the method comprising the step of parenterallyadministering to the vertebrate subject an immunologically effectiveamount of a) a parenteral adjuvant comprising a polynucleotide encodinga detoxified mutant of an E. coli heat-labile (LT) ADP-ribosylatingtoxin in combination with a pharmaceutically acceptable vehicle, whereinsaid detoxified mutant is LT-K63; and b) at least one selected antigen.2. The method according to claim 1, wherein the adjuvant and the antigenare administered subcutaneously, transcutaneously or intramuscularly. 3.The method according to claim 1, wherein the pharmaceutically acceptablevehicle is a topical vehicle.
 4. The method according to claim 1,wherein the adjuvant and the antigen are administered transcutaneously.5. The method according to claim 1, wherein the adjuvant is administeredto the vertebrate subject prior to administering the selected antigen.6. The method according to claim 1, wherein the adjuvant is administeredto the vertebrate subject subsequent to administering the selectedantigen.
 7. The method according to claim 1, wherein the antigen isadministered to the vertebrate subject concurrent with administering theselected antigen.
 8. The method of claim 1, wherein said antigen is aviral antigen.
 9. The method of claim 8, wherein said viral antigen isselected from the group consisting of an influenza antigen, a herpessimplex virus (HSV) antigen, a human immunodeficiency virus (HIV)antigen, a cytomegalovirus (CMV) antigen, a hepatitis C virus (HCV)antigen, a delta hepatitis virus (HDV) antigen, a poliovirus antigen, ahepatitis A virus (HAV) antigen, an Epstein-Barr virus (EBV) antigen, avaricella zoster virus (VZV) antigen, and a respiratory syncytial virus(RSV) antigen.
 10. The method of claim 9, wherein said viral antigen isan influenza virus antigen.
 11. The method of claim 9, wherein saidviral antigen is a poliovirus antigen.
 12. The method of claim 9,wherein said viral antigen is a RSV antigen.
 13. The method of claim 1,wherein said antigen is a bacterial antigen.
 14. The method of claim 13,wherein said bacterial antigen is selected from the group consisting ofBordetella pertussis antigens, Helicobacter pylori antigens,meningococcus A antigens, meningococcus B antigens, and meningococcus Cantigens.
 15. The method of claim 14, wherein said bacterial antigen isa Bordetella pertussis antigen.
 16. The method of claim 14, wherein saidbacterial antigen is an Helicobacter pylori antigen.
 17. The method ofclaim 14, wherein said bacterial antigen is a meningococcus A antigen.18. The method of claim 14, wherein said bacterial antigen is ameningococcus B antigen.
 19. The method of claim 14, wherein saidbacterial antigen is a meningococcus C antigen.